Several publications and patent documents are referenced in this application in order to more fully describe the state of the art to which this invention pertains. The disclosure of each of these publications and documents is incorporated by reference herein.
Extracellular signaling molecules bound to cell surface receptors can regulate nuclear function with consequences for cell proliferation, differentiation, and function. To regulate nuclear function, signals must be transduced across the nuclear envelope to propagate the signal from the cytoplasm to the nucleus. Many signaling responses induce the nuclear translocation of transcription factors, kinases and others (Cyert. (2001) J. Biol. Chem. 276, 20805-20808; Brivanlou et al. (2002) Science 295, 813-818).
Diverse mechanisms exist that regulate how these molecules are activated to move or translocate into and out of the nucleus. Phosphorylation is one of the most extensively characterized post-translational modifications that contributes to the ability of molecules to undergo nuclear translocation. In signal transduction from type I and type II cytokine receptors, for example, signal transducer and activator of transcription (Stat) proteins, which usually reside within the cytoplasm, become activated upon selective phosphorylation of tyrosine residues by Jak protein tyrosine kinases. Phosphorylated Stat proteins subsequently form complexes (e.g., homodimers), thus becoming active transcription factors that translocate into the nucleus to regulate transcription of their target genes (Ihle (1996) Cell 84, 331-334; Stark et al. (1998) Annu. Rev. Biochem. 67, 227-264; Levy et al. (2002) Nat. Rev. Mol. Cell Biol. 3, 651-662). Serine phosphorylation also plays an important role in nuclear translocation of Smad transcription factors in transforming growth factor-β (TGFβ) signaling (Massagué. (2000) Nat. Rev. Mol. Cell Biol. 1, 169-178). In addition to transcription factors, other signaling molecules, such as mitogen-activated protein kinase, also translocate into the nucleus upon targeted phosphorylation (Chen et al. (1992) Mol. Cell. Biol. 12, 915-927).
Calcium signaling, however, activates the phosphatase calcineurin, which in turn dephosphorylates and induces nuclear localization of the cytoplasmic components of nuclear factor of activated T cells (NFATc) (Crabtree et al. (2002) Cell 109, S67-S79). In contrast, lipopolysaccharide and inflammatory cytokines activate nuclear factor κB (NF-κB) transcription factor to move into the nucleus by the targeted phosphorylation and subsequent degradation of IκB (Baeuerle et al. (1996) Cell 87, 13-20; Baldwin. (1996) Annu. Rev. Immunol. 14, 649-681). Proteolytic cleavage is used differently for signal-induced nuclear translocation as revealed in activation of sterol regulatory element binding proteins (SREBP) in cholesterol metabolism (Horton et al. (2002) J. Clin. Invest. 109, 1125-1131) or in Notch signaling (Schroeter et al. (1998) Nature 393, 382-386; Struhl et al. (1998) Cell 93, 649-660). These diverse mechanisms of regulated nuclear translocation have made it difficult to devise general methods to identify proteins whose translocation into the nucleus is induced by extracellular stimuli.
As described herein below, the present invention addresses the need for an assay that can identify proteins whose translocation into the nucleus is induced by extracellular stimuli.